Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments

Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over‐represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over‐represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate‐reducing bacteria and sulphur‐oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico‐chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.     

Analysing diversity and community structures using PCR‐RFLP: a new software application

Restriction fragment length polymorphism tools is an R application which supports a complete workflow of polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), dealing with the problems which accompany analysis when PCR‐RFLP is used in diversity studies. Large numbers of different RFLP samples obtained from multiple electrophoresis runs might lead to limitations or misidentifications due to the need for band matching in most existing software applications. Due to the common problem of variation in the density of bands (i.e. distances between bands or visual intensity) in the electropherograms, it is desirable to have options for handling samples with uncertain or faint bands. As a further step in the workflow, scientists often use DNA sequencing to identify individual genotypes, so that the use of specific software to combine these tasks might be helpful. With this background, we here present an application that supports a complete workflow, starting with the analysis of single species samples by PCR‐RFLP, to PCR‐RFLP genotype identification based on a reference data set and DNA sequencing followed by similarity analysis. RFLPtools is a freely available, platform‐independent application which provides analysis functions for DNA fragment molecular weights (e.g. by RFLP analysis), including similarity calculations without the need for band matching. As it is written for the statistical software R, other statistical analyses might also be easily applied.     

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

Is photoisomerization required for NO photorelease in ruthenium nitrosyl complexes?

The factors that explain the competition between intramolecular NO linkage photoisomerization and NO photorelease in five ruthenium nitrosyl complexes were investigated. By applying DFT-based methods, it was possible to characterize the ground states and lowest triplet potential energy surfaces of these species, and to establish that both photoisomerization and photorelease processes can occur in the lowest triplet state of each species. This work highlights the crucial role of the sideways-bonded isomer, a metastable state also known as the MS2 isomer, in the photochemical loss of NO, while the results obtained also indicate that the population of the triplet state of this isomer is compulsory for both processes and show how photoisomerization and photorelease interfere.

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Graphical Abstract Illustration of the crucial role of the ³MS2 state in the photoreactivities of ruthenium nitrosyl complexes

     

B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

Prolonged Cerebral Circulation Time Is the Best Parameter for Predicting Vasospasm during Initial CT Perfusion in Subarachnoid Hemorrhagic Patients

Purpose

We sought to imitate angiographic cerebral circulation time (CCT) and create a similar index from baseline CT perfusion (CTP) to better predict vasospasm in patients with subarachnoid hemorrhage (SAH).

Methods

Forty-one SAH patients with available DSA and CTP were retrospectively included. The vasospasm group was comprised of patients with deterioration in conscious functioning and newly developed luminal narrowing; remaining cases were classified as the control group. The angiography CCT (XA-CCT) was defined as the difference in TTP (time to peak) between the selected arterial ROIs and the superior sagittal sinus (SSS). Four arterial ROIs were selected to generate four corresponding XA-CCTs: the right and left anterior cerebral arteries (XA-CCT_RA2 and XA-CCT_LA2) and right- and left-middle cerebral arteries (XA-CCT_RM2 and XA-CCT_LM2). The CCTs from CTP (CT-CCT) were defined as the differences in TTP from the corresponding arterial ROIs and the SSS. Correlations of the different CCTs were calculated and diagnostic accuracy in predicting vasospasm was evaluated.

Results

Intra-class correlations ranged from 0.96 to 0.98. The correlations of XA-CCT_RA2, XA-CCT_RM2, XA-CCT_LA2, and XA-CCT_LM2 with the corresponding CT-CCTs were 0.64, 0.65, 0.53, and 0.68, respectively. All CCTs were significantly prolonged in the vasospasm group (5.8–6.4 s) except for XA-CCT_LA2. CT-CCT_A2 of 5.62 was the optimal cut-off value for detecting vasospasm with a sensitivity of 84.2% and specificity 82.4%

Conclusion

CT-CCTs can be used to interpret cerebral flow without deconvolution algorithms, and outperform both MTT and TTP in predicting vasospasm risk. This finding may help facilitate management of patients with SAH.     

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.     

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) ‘Bright Yellow 2’ cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.Eukaryotic plasma membranes (PMs) are composed of three main classes of lipids, glycerolipids, sphingolipids, and sterols, which may account for up to 100,000 different molecular species (Yetukuri et al., 2008; Shevchenko and Simons, 2010). Overall, all glycerolipids share the same molecular moieties in plants, animals, and fungi. By contrast, sterols and sphingolipids are different and specific to each kingdom. For instance, the plant PM contains an important number of sterols, among which β-sitosterol, stigmasterol, and campesterol predominate (Furt et al., 2011). In addition to free sterols, phytosterols can be conjugated to form steryl glycosides (SG) and acyl steryl glycosides (ASG) that represent up to approximately 15% of the tobacco (Nicotiana tabacum) PM (Furt et al., 2010). As for sphingolipids, sphingomyelin, the major phosphosphingolipid in animals, which harbors a phosphocholine as a polar head, is not detected in plants. Glycosyl inositol phosphorylceramides (GIPCs) are the major class of sphingolipids in plants, but they are absent in animals (Sperling and Heinz, 2003; Pata et al., 2010). Sphingolipidomic approaches identified up to 200 plant sphingolipids (for review, see Pata et al., 2010; Cacas et al., 2013).Although GIPCs belong to one of the earliest classes of plant sphingolipids that were identified in the late 1950s (Carter et al., 1958), only a few GIPCs have been structurally characterized to date because of their high polarity and a limited solubility in typical lipid extraction solvents. For these reasons, they were systematically omitted from published plant PM lipid composition. GIPCs are formed by the addition of an inositol phosphate to the ceramide moiety, the inositol headgroup of which can then undergo several glycosylation steps. The dominant glycan structure, composed of a hexose-GlcA linked to the inositol, is called series A. Polar heads containing three to seven sugars, so-called series B to F, have been identified and appeared to be species specific (Buré et al., 2011; Cacas et al., 2013; Mortimer et al., 2013). The ceramide moiety of GIPCs consists of a long-chain base (LCB), mainly t18:0 (called phytosphingosine) or t18:1 compounds (for review, see Pata et al., 2010), to which is amidified a very-long-chain fatty acid (VLCFA), the latter of which is mostly 2-hydroxylated (hVLCFA) with an odd or even number of carbon atoms. In plants, little is known about the subcellular localization of GIPCs. It is assumed, however, that they would be highly represented in the PM (Worrall et al., 2003; Sperling et al., 2005), even if this remains to be experimentally proven. The main argument supporting such an assumption is the strong enrichment of trihydroxylated LCB (t18:n) in detergent-insoluble membrane (DIM) fractions (Borner et al., 2005; Lefebvre et al., 2007), LCB being known to be predominant in GIPC’s core structure as aforementioned.In addition to this chemical complexity, lipids are not evenly distributed within the PM. Sphingolipids and sterols can preferentially interact with each other and segregate to form microdomains dubbed the membrane raft (Simons and Toomre, 2000). The membrane raft hypothesis suggests that lipids play a regulatory role in mediating protein clustering within the bilayer by undergoing phase separation into liquid-disordered and liquid-ordered phases. The liquid-ordered phase, termed the membrane raft, was described as enriched in sterol and saturated sphingolipids and is characterized by tight lipid packing. Proteins, which have differential affinities for each phase, may become enriched in, or excluded from, the liquid-ordered phase domains to optimize the rate of protein-protein interactions and maximize signaling processes. In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (Simons and Ikonen, 1997; Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see Mongrand et al., 2010; Simon-Plas et al., 2011; Cacas et al., 2012a).Moreover, lipids are not evenly distributed between the two leaflets of the PM. Within the PM of eukaryotic cells, sphingolipids are primarily located in the outer monolayer, whereas unsaturated phospholipids are predominantly exposed on the cytosolic leaflet. This asymmetrical distribution has been well established in human red blood cells, in which the outer leaflet contains sphingomyelin, phosphatidylcholine, and a variety of glycolipids like gangliosides. By contrast, the cytoplasmic leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and their phosphorylated derivatives (Devaux and Morris, 2004). With regard to sphingolipids and glycerolipids, the asymmetry of the former is established during their biosynthesis and that of the latter requires ATPases such as the aminophospholipid translocase that transports lipids from the outer to the inner leaflet as well as multiple drug resistance proteins that transport phosphatidylcholine in the opposite direction (Devaux and Morris, 2004). This ubiquitous scheme encountered in animal cells could apply in plant cells as proposed (Tjellstrom et al., 2010). Indeed, the authors showed that there is a pronounced transverse lipid asymmetry in root at the PM. Phospholipids and galactolipids dominate the cytosolic leaflet, whereas the apoplastic leaflet is enriched in sphingolipids and sterols.From such a high diversity of the plant PM thus arises the question of the respective contribution of lipids to membrane suborganization. Our group recently tackled this aspect by characterizing the order level of liposomes prepared from various plant lipids and labeled with the environment-sensitive probe di-4-ANEPPDHQ (Grosjean et al., 2015). Fluorescence spectroscopy experiments showed that, among phytosterols, campesterol exhibits the strongest ability to order model membranes. In agreement with these data, spatial analysis of the membrane organization through multispectral confocal microscopy pointed to the strong ability of campesterol to promote liquid-ordered domain formation and organize their spatial distribution at the membrane surface. Conjugated sterols also exhibit a striking ability to order membranes. In addition, GIPCs enhance the sterol-induced ordering effect by emphasizing the formation and increasing the size of sterol-dependent ordered domains.The aim of this study was to reinvestigate the lipid composition and organization of the PM with a particular focus on GIPCs using tobacco leaves and cv Bright Yellow 2 (BY-2) cell cultures as models. Analyzing all membrane lipid classes at once, including sphingolipids, is challenging because they all display dramatically different chemical polarity, from very apolar (like free sterols) to highly polar (like polyglycosylated GIPCs) molecules. Most lipid extraction techniques published thus far use a chloroform/methanol mixture and phase partition to remove contaminants, resulting in the loss GIPCs, which remain in the aqueous phase, unextracted in the insoluble pellet, or at the interphase (Markham et al., 2006). In order to gain access to both glycerolipid and sphingolipid species at a glance, we developed a protocol whereby the esterifed or amidified fatty acids were hydrolyzed from the glycerol backbone (glycerolipids) or the LCB (sphingolipids) of membrane lipids, respectively. Fatty acids were then analyzed by gas chromatography-mass spectrometry (GC-MS) with appropriate internal standards for quantification. We further proposed that the use of methyl tert-butyl ether (MTBE) ensures the extraction of all classes of plant polar lipids. Our results indicate that GIPCs represent up to 40 mol % of total tobacco PM lipids. Interestingly, polyglycolyslated GIPCs are 5-fold enriched in DIMs of BY-2 cells when compared with the PM. Further investigation led us to develop a preparative purification procedure that allowed us to obtain enough material to raise antibodies against GIPCs. Using immunogold labeling on PM vesicles, it was found that polyglycosylated GIPCs cluster in membrane nanodomains, strengthening the idea that lateral nanosegregation of sphingolipids takes place at the PM in plants. Multispectral confocal microscopy was performed on vesicles prepared using GIPCs, phospholipids, and sterols and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Our results show that, despite different fatty acid and polar head compositions, GIPCs extracted from tobacco leaves and BY-2 cells have a similar intrinsic propensity of enhancing vesicle global order together with sterols. Assuming that GIPCs are mostly present in the outer leaflet of the PM, interactions between sterols and sphingolipids were finally studied by the Langmuir monolayer technique, and the area of a single molecule of GIPC, or in interaction with phytosterols, was calculated. Using the calculation docking method, the energy of interaction between GIPCs and phytosterols was determined. A model was proposed in which GIPCs and phytosterols interact together to form liquid-ordered domains and in which the VLCFAs of GIPCs promote the interdigitation of the two membrane leaflets. The implications of domain formation and the asymmetrical distribution of lipids at the PM in plants are also discussed. Finally, we propose a model that reconsiders the intricate organization of the plant PM bilayer.